Anna was using the Nikon microscope a couple of days ago, and noticed some level of bleed-over of mCherry fluorescence into the CY5 filter-set channel. I previously played around with making a bespoke script for figuring out the optimal fluorescent protein : laser : detector combinations on the two flow cytometers we routinely use at the core here, and figured I could modify the script to understand how our existing Nikon filter sets work with the various fluorescent proteins we use here. It took me a couple hours, but I made a script posted on my GitHub. Here’s a figure that script produces:

Based on the filter sets we currently have, looks like it may be near impossible to avoid bleedover of a near infra-red FP fluorescence into the red channel. Also, this seems to reproduce / explain the effect Anna was seeing with mCherry fluorescence bleedover into the NIR channel. Seems this problem doesn’t happen with mScarlet.I, so maybe I’ll slowly shift toward using that FP more.